Trinucleotide repeat sequences (TNRs), such as CAG/CTG repeats, expand in the human genome to cause 17 inherited human diseases, including Huntington's disease, myotonic dystrophy, and spinocerebellar ataxias. The strong hairpin-forming potential of these sequences is the likely basis for genome instability at expanded TNRs. Expanded TNRs inhibit repair of DNA gaps, interfere with DNA replication, and cause chromosomes to break (chromosome fragility). The goal of our research is to elucidate the cellular mechanisms that normally facilitate replication and repair of TNRs and determine how failure of these systems can lead to repeat expansion and chromosome breakage. We propose to characterize the role of proteins involved in DNA repair, the DNA damage checkpoint, and chromatin modification in preventing CAG/CTG repeat fragility and expansion, using Saccharomyces cerevisiae as a model system. The repair response to naturally occurring damage at strong hairpin-forming sequences will be investigated by using chromatin immunoprecipitation and fluorescence techniques to determine the identity and timing of repair protein recruitment to expanded TNRs. To establish the cellular consequences of DNA damage checkpoint activation by TNRs, division of single cells containing long CAG/CTG repeats will be monitored, and the role of the DNA damage checkpoint in preventing replication fork stalling at long repeats ascertained by 2-D gels. A novel link between TNR stability and chromatin structure will be investigated by determining the state of histone modifications and spacing at expanded TNRs. Lastly, we propose a genetic screen to identify additional proteins and pathways important in preventing chromosome fragility at CAG/CTG tracts. An understanding of how repair of CAG/CTG sequences occurs is important both to explain how repeat expansion diseases occur and to prevent pathological somatic expansions in non-dividing brain cells in Huntington's disease. Since cell death is a major factor in progression of several TNR diseases, determining the consequences of damage at expanded repeats for cell health could lead to strategies to slow disease development. The human genome contains many direct and inverted repeats and other types of sequences that present potential problems during replication and repair, so understanding the cellular mechanisms that have evolved to cope with these problems should yield important insights into genome stability.